The present invention relates to a process for expression of protein products in Aspergillus, recombinant DNA vectors, a promoter for Aspergillus and transformed fungi. The present invention is also directed to a new amylase from A. niger.
In the past, numerous processes have been developed for the production of polypeptides or proteins by means of the recombinant DNA technology. The main interest has been concentrated on bacteria and yeast, e.g. E. coli, Bacillus subtilis and Saccharomyces cerevisiae being well characterized species as regards for instance expression and selection systems.
Besides the above mentioned microorganisms, filamentous fungi, such as Aspergillus niger, are attractive candidates as host microorganisms for recombinant DNA vectors being well-characterized and widely used microorganisms for the commercial production of enzymes. Efforts have especially been concentrated on the development of transformation systems by which a selection marker permitting selection of transformants from the untransformed host microorganisms is used.
In the last few years different selection markers for the transformation of Aspergillus nidulans have been described and procedures have been developed for integrative transformation of the filamentous fungus Aspergillus nidulans for the purpose of investigation of the genetic and molecular processes controlling fungal cell differentiation.
Transformation of A. nidulans has been demonstrated by using plasmids containing the Neurospora crassa pyr-4 gene (Ballance, D. J. et al., Biochem. Biophys. Res. Commun , 112 (1983) 284-289), the A. nidulans amdS gene (Tilburn, J. G. et al., Gene 26 (1983) 205-221), the A. nidulans trpC gene (Yelton, M. M. et al., Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1470-1474) and the A. nidulans argB gene (John, M. A. and Peberdy J., Microb. Technol. 6 (1984) 386-389). The transforming DNA was found to be integrated into the host genome at rather low frequencies (typically &lt;1000 transformants/.mu.g of DNA).
Recently transformation of Aspergillus niger with the amdS gene of A. nidulans was described (Kelly, J. M. and Hynes, M. J., EMBO Journal 4 (1985), 475-479) and amdS was shown to be a potential selection marker for use in transformation of Aspergillus niger that cannot grow strongly on acetamide as a sole nitrogen source. Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans has also been described recently (Buxton, F. P. et al., Gene 37 (1985), 207-214).
So far yields of heterologous proteins have not been satisfactory in A. niger for commercial production. Accordingly, it is the object of the present invention to provide a method for obtaining commercially attractive yields of foreign proteins in Aspergillus. It is also an object of the present invention to enhance the production of homologous proteins in Aspergillus.